Spermatogenesis creates functional sperm from an initially undifferentiated germ cell.
In the nematodeCaenorhabditis elegans,both males and
hermaphrodites engage in spermatogenesis. The hermaphrodite germ line, like that of the
male, initiates spermatogenesis during the L4 larval stage. The hermaphrodite germ line
differs from that of the male because it ceases spermatogenesis and switches to
oogenesis during the adult stage. Each hermaphrodite stores her sperm and uses them to
fertilize her oocytes. Many mutants have been identified where hermaphrodite
self-fertility is disrupted. If such a self-sterile hermaphrodite is mated to a
wild-type male, mutant hermaphrodites that either lack sperm or contain defective sperm
will produce outcross progeny. Easily implemented tests are then applied to identify the
subset of these mutants that produce defective sperm. Currently, more than 44 genes are
known that are required for normal spermatogenesis. This chapter discusses the 25
best-understood genes that affect spermatogenesis and mutants are grouped based on the
cellular structure or process that is affected.C. elegansspermatozoa lack an acrosome and a flagellum, which are organelles found in the
spermatozoa produced by most other species. Like other nematodes,C.
elegansspermatozoa move by crawling using a single pseudopod. Wild-type
spermatogenesis and its defects in mutants can be studiedin vivobecause the animal is transparent andin vitrobecause a simple,
chemically defined medium that supports development has been discovered. Unlike nearly
all otherC. eleganscells, homogeneous sperm can be obtained in
sufficient quantities to permit biochemical analyses.

2. Wild-type spermatogenesis

Development of sperm in C. elegans males has been described in
detail (Kimble and Ward, 1988; Ward, 1986; Ward et al., 1981;
Wolf et al., 1978) and these cellular events are reviewed in Figure 1. The primary spermatocyte initially forms in syncytium with a
cytoplasmic core called the rachis. Upon entering meiosis, the primary spermatocyte buds
off the rachis and completes development without any requirement for intimate
association with other cells. As in most cells, nuclear divisions are closely
coordinated with cytokinesis and other aspects of cytodifferentiation. Meiosis I divides
the 4N primary spermatocyte nucleus into two 2N nuclei in secondary spermatocytes. The
cytokinesis that accompanies meiosis I is either complete (not shown) or partial (2 in
Figure 1A). Either way, the resulting cells are secondary
spermatocytes and they immediately initiate meiosis II. Meiosis II produces two haploid
spermatids from each 2N secondary spermatocyte. Spermatids form by budding from an
anucleate residual body (Movie 1), and spermatids lack many
cellular constituents present in the secondary spermatocyte. Material within the
residual body, which includes most voltage-gated ion channels, tubulin, actin and all
ribosomes (Machaca et al., 1996; Ward, 1986) is apparently resorbed
(Kimble and Ward, 1988; Nelson et al., 1982; Ward, 1986; Ward et al., 1981; Wolf et al., 1978). Sessile spermatids lack
ribosomes and have no new protein synthesis as they complete differentiation into motile
spermatozoa (Ward et al., 1983). The cytologically obvious structures that segregate
into spermatids during budding include its nucleus, multiple mitochondria and multiple
specialized lysosome-like organelles named fibrous body-membranous organelles (FB-MOs).

The meiotic divisions of spermatogenesis are coordinated with the morphogenesis of
FB-MOs (Ward et al., 1981; Wolf et al., 1978). In syncytial
pachytene spermatocytes (Figure 1), the MOs form from the Golgi
apparatus and FBs form in close association with the MOs (Roberts et al., 1986). Growth of the FBs occurs within a MO-derived membrane envelope in primary
spermatocytes (Figure 1B1). As spermatids bud, the double membrane
surrounding the FB retracts into the MO and the filamentous major sperm protein (MSP)
contents (Figure 1B3) depolymerize and disperse throughout the
spermatid cytoplasm. These FB-free MOs (Figure 1B4) localize near the
spermatid plasma membrane (pm in Figure 1B4). Spermiogenesis is the
process during which a spermatid becomes a spermatozoon. MO head membranes (h, in Figure 1B4) fuse and deposit their contents on the plasma membrane of the
cell body as the spermatozoon forms (Figure 1B5). A single pseudopod is
extended (red arrows at 3 in Figure 1A), and MSP fibers are confined to
the pseudopod (Ward and Klass, 1982) while the nucleus, mitochondria, MOs
and other internal membranes are confined to the cell body. Like other nematodes,
C. elegans spermatozoa lack a flagellum and crawl using this
single pseudopod (see Sperm motility and MSP).

Figure 1. (A). Summary of spermatogenesis. Asymmetric partitioning of cellular constituents
occurs at three points during C. elegans spermatogenesis, as indicated by double-headed
red arrows at the red numbers. 1, syncytial pachytene spermatocytes with many FB-MOs bud
from the rachis and divide to form secondary spermatocytes (FB-MOs are shown in green);
2, spermatids selectively retain FB-MOs as they bud from the residual body; 3, FB-MOs
fuse with the spermatid plasma membrane as a pseudopod extends from the cell body during
spermiogenesis. Nuclei are the circles in the center of each cell. Nuclei are patterned
with lines to represent stages when chromatin is in condensed meiotic chromosomes or
filled (in black) after chromatin forms a single highly condensed sphere. (B) Summary of
morphogenesis of the FB-MO complex. 1, The fibrous body (FB) develops in close
association with, and is surrounded by, the membranous organelle (MO) within the primary
spermatocyte. The MO is separated by a collar region (c) into a head (h; speckled region
at left) and body (b; region to the right of the collar); 2, the FB-MO complex reaches
its largest size within primary spermatocytes. The double layered MO-derived membrane
surrounds the FB, which is composed of the major sperm protein filaments; 3, the
MO-derived membranes surrounding the FB retract and fold up while FB filaments
depolymerize and disperse as spermatids bud from the residual body; 4, the head of each
MO (arrow) moves to a position just below the plasma membrane (pm) of the spermatid
after the FBs have depolymerized and disappeared. The irregular shapes within the FB
represent retracted membrane that had covered the exterior of the FB; 5, the head of the
MO fuses at the collar to the plasma membrane and exocytoses its contents (dots at
arrow) onto the cell surface. A permanent fusion pore remains at the point of each MO
fusion (each cell has many MO's; B is modified from L'Hernault and Arduengo, 1992).

Figure 2. Video micrograph of spermatid budding. Two connected secondary spermatocytes form four spermatids and place much of the
cytoplasm in the centrally located residual body. QuickTime movie courtesy of P. J. Muhlrad and S. Ward, University
of Arizona.

Spermatogenesis is similar in hermaphrodites and males, but there are some significant
differences. Spermatids accumulate in the proximal gonad arm after the hermaphrodite
germ line has switched to oogenesis (Figure 3A). The first ovulation
pushes spermatids into the spermatheca (Figure 3B), where they rapidly
undergo spermiogenesis and become spermatozoa (Figure 3C). Many
spermatozoa are displaced into the uterus as the egg leaves the spermatheca and they
must crawl back into the spermatheca in order to compete for oocytes (Figure 3D-F; Ward and Carrel, 1979).

Figure 3. Summary of gamete interactions in the hermaphrodite during self-fertilization. (A) Spermatids (in pink) are crowded at the oviduct spermathecal junction by maturing
oocytes. (B) Spermatids are pushed into the spermatheca by the first ovulation. (C)
Spermatids undergo spermiogenesis to become spermatozoa and compete with each other to
fertilize the oocyte. (D) The newly fertilized egg exits the spermatheca and pushes many
sperm into the uterus. (E) The displaced sperm crawl from the uterus back into the
spermatheca. (F) The second ovulation places an oocyte in the spermatheca and sperm
compete to fertilize it. The cell body is pulled behind the pseudopod as spermatozoa
crawl forward.

In males, spermatids are stored in the seminal vesicle. These spermatids are
significantly larger than those produced by hermaphrodites (LaMunyon and Ward, 1998). Spermatids undergo spermiogenesis after they are mixed with seminal fluid
during ejaculation into the hermaphrodite uterus. The molecular mechanism that activates
spermiogenesis in vivo is not known for either sex. However,
in vitro, spermatids are activated to become spermatozoa by
treatment with several unrelated agents including the ionophore monensin (Nelson and Ward, 1980), proteases, weak bases that alkalinize the cytoplasm (Ward et al., 1983), calmodulin inhibitors (Shakes and Ward, 1989) or
the ion channel inhibitor DIDS (Machaca et al., 1996). Normally,
male-derived spermatozoa crawl towards and into the hermaphrodite spermatheca.
Spermatozoa introduced by a male during copulation have a competitive advantage over
hermaphrodite-derived spermatozoa in the struggle to fertilize oocytes (Figure 4; Ward and Carrel, 1979; LaMunyon and Ward, 1995). Artificial insemination is also possible, and male-derived spermatids that are
protease treated cannot fertilize oocytes, while spermatids treated with weak bases are
competent to participate in fertilization (LaMunyon and Ward, 1994).

Figure 4. Sperm competition. (A) Hermaphrodite-derived spermatozoa wait in the spermatheca, where they fertilize
nearly all oocytes that pass through the spermatheca. (B) A hermaphrodite after being
inseminated by relatively few male-derived (blue) sperm will produce a mixture of self
(pink embryo) and outcross (blue embryo) progeny. (C) A hermaphrodite after being
inseminated by many male-derived (blue) sperm will produce only outcross (blue embryo)
progeny. As noted by others, male-derived sperm are ~50% larger than
hermaphrodite-derived sperm (LaMunyon and Ward, 1995; LaMunyon and Ward, 1998). Adapted from Singson et al. (1999).

3. Identification of spermatogenesis defective mutants

C. elegans spermatogenesis mutants (spe or
fer) have been identified because they compromise hermaphrodite
self-fertility. In wild-type, virtually every sperm fertilizes an oocyte in young
hermaphrodites, and the resulting shelled, oval eggs are laid on the agar growth plate
(Ward and Carrel, 1979). In contrast, spe/fer mutants
lay unfertilized oocytes, which are round, brown cells that lack an eggshell. The
differences between laid oocytes and eggs are easily discerned under the dissecting
microscope. Mutant, self-sterile hermaphrodites that have a normal appearing germ line
can be readily identified following mutagenesis. While some of these mutants never
initiate spermatogenesis (see Sex determination in the germ line), most produce defective sperm. While mutants that fail to
initiate spermatogenesis lay a few oocytes, mutants that make defective sperm lay large
numbers of unfertilized oocytes (McCarter et al., 1999). Self-sterile
hermaphrodites can be mated to wild-type males, allowing the recovery of a spe or fer
mutation among the outcross progeny. This straightforward screen has allowed
identification of > 44 genes that affect
spermatogenesis (reviewed by L'Hernault, 1997; L’Hernault and Singson, 2000;
S. L’Hernault, unpublished; S. Ward, unpublished) and several additional genes were
identified in other studies (Amiri et al., 2001; Luitjens et al., 2000; Subramaniam and Seydoux, 2003); representative mutants are
shown in Figure 5.

Figure 5. (A) Stages of wild type spermatogenesis are shown diagramatically as an ordered
pathway of morphogenesis with the stages labeled in blue. Some of the > 44 known genes are placed on the pathway where
ultrastructural or light microscopic defects are evident. The ife-1
gene is not shown, but it slows down the rate of spermatogenesis and also affects
spermatozoa. The next to last step of the pathway represents mutant spermatozoa that are
cytologically normal but cannot enter oocytes. The spe-11 mutant is
a paternal effect lethal mutant that makes spermatozoa that enter the egg and the
resulting defective embryo dies. (B) The most common terminal stages of mutants that
arrest morphogenesis without forming spermatids. (C) The protein distribution of SPE-9
(arrows), the hypothesized SPE-9 receptor (“Y” on egg surface) and the SPE-41/TRP-3
protein (red dots). Please note that individual drawings are not to scale; for instance,
spermatozoa are 5–6 μm in diameter while
the eggs shown are ~45 μm (modified from L'Hernault et al., 1988).

4. Translational control during spermatogenesis

Translational control is widely employed in differentiating cellular systems (reviewed
by Gebauer and Hentze, 2004), and two genes have been identified that act
as spermatogenesis-specific regulators of translation.

The cpb-1 gene encodes a cytoplasmic polyadenylation element binding protein. Although
this gene is expressed in both the testes and the ovary, RNAi experiments reveal
that it is required for spermatogenesis but not oogenesis.
cpb-1(RNAi) hermaphrodites lay unfertilized oocytes, like
spe/fer mutants. Examination of the spermatheca reveals
that cpb-1(RNAi) hermaphrodites accumulate spermatocytes
and many of these cells fail to complete meiosis. These results suggest that
cpb-1 regulates the translation of one or more mRNAs that
are critical for completion of meiosis and perhaps other processes during
spermatogenesis (Luitjens et al., 2000).

The ife-1 gene encodes an eIF4E mRNA cap binding protein that is associated with P
granules during germline proliferation. RNAi of the ife-1
gene results in a temperature sensitive delay in the completion of
spermatogenesis, but the majority of the resulting sperm are not competent to
engage in fertilization. About 80% of ife-1(RNAi) treated
hermaphrodites are self-sterile and the remaining 20% have reduced broods. These
data suggest that IFE-1 regulates translation of one or more RNA that are
critical for spermatogenesis and sperm function (Amiri et al., 2001).

5. Mutants that affect sperm meiosis

Like other animals, C. elegans meiosis occurs during both
oogenesis and spermatogenesis, and many meiotic processes are similar in the two germ
lines. However, two genes that are
involved in meiosis can show specific defects during spermatogenesis.

Six dominant wee-1.3(gf) Spe mutants have been discovered and they have
no evident defects during oogenesis or in any proliferating somatic tissue. This
observation is surprising because Wee1p type kinase is widely expressed (A.
Golden, personal communication; Lamitina and L'Hernault, 2002)
and probably negatively regulates the C. eleganscdc2p ortholog ncc-1 (Boxem et al., 1999); cdc2p (or its orthologs) is a master
regulator of both mitotic and meiotic divisions in eukaryotes (Lundgren et al., 1991). The wee-1.3 null phenotype is
embryonic/larval lethality, which indicates it plays an essential role in
addition to its role during spermatogenesis (Lamitina and L'Hernault, 2002). These independently obtained dominant wee-1.3
mutations are all located in a four amino acid region near the C-terminus and
three contain identical mutations. Dominant wee-1.3 mutant
spermatocytes enter pachytene of meiosis I, but the nuclear envelope remains
intact and the chromatin acquires an ultrastructural appearance that is similar
(although ~4X larger) to that seen in wild-type (haploid) spermatids.
wee-1.3 dominant mutants do not initiate cytokinesis and
they arrest with an undivided nucleus on one side of the primary spermatocyte
(Figure 5B) and vacuolated FB-MOs on the opposite side.
These data indicate that a sperm-specific pathway negatively regulates WEE-1.3
during the G2/M transition of male meiosis I, and this regulation does not occur
in these dominant mutants (Lamitina and L'Hernault, 2002).

puf-8 encodes a pumilio-like RNA binding protein that
controls RNA stability and translation. The PUF-8 protein is required
redundantly with other PUF proteins to maintain viable germ cells during
development (Subramaniam and Seydoux, 1999), but it also plays a
partially penetrant, non redundant role in the testes. Primary spermatocytes
lacking PUF-8 complete prophase of meiosis I, but then exit meiosis, re-enter
mitosis and de-differentiate into tumorous germ cells. This observation
indicates that PUF-8 is required for primary spermatocytes to complete meiosis
and remain in the spermatogenesis pathway (Subramaniam and Seydoux, 2003).

6. Mutants affecting FB-MOs

Several mutants affect FB-MO morphogenesis or function.

The spe-39
gene encodes a novel hydrophilic, cytoplasmic protein that is not specifically
associated with FB-MOs in spermatocytes and spermatids.
spe-39 mutants arrest as aberrant spermatocytes (Figure 5B) that lack MOs but contain many small vesicles with
internal membranes, a distinctive feature of wild-type MOs (Figure 1). A membrane envelope does not surround
spe-39 FBs, indicating that some aspects of the FB can form
in the absence of MOs. In addition to its role during spermatogenesis, SPE-39 is
found in many, if not all, C. elegans cells, and
spe-39 RNAi causes embryonic lethality. The
spe-39 gene has orthologs in many metazoa, but no homolog
was identified in the yeast genome. This observation suggests that the SPE-39
role during FB-MO morphogenesis is a conserved process that requires SPE-39
orthologs in other metazoan cell types (Zhu and L'Hernault, 2003).

The spe-6
gene encodes a casein I type serine threonine kinase (Muhlrad and Ward, 2002). In spe-6 null mutants, the MSP fails to
form FBs (Varkey et al., 1993). Consequently, FB-MOs never form,
so spe-6 mutants do not form spermatids and arrest as
aberrant spermatocytes (Figure 5B). This observation suggests
that a regulatory phosphorylation controls FB morphogenesis, but the substrates
for the SPE-6 kinase are not currently defined. Non-null mutants reveal that
this gene is also involved in regulating spermiogenesis (see Sex-specific aspects of spermiogenesis, below).

The spe-4
gene encodes a sperm-specific presenilin (L'Hernault and Arduengo, 1992); presenilins are intramembranous aspartyl proteases that process other
membrane proteins, including the Alzheimer precursor protein in human brain (Xia and Wolfe, 2003). The SPE-4 protein resides in FB-MOs and
spe-4 mutants, like spe-39 mutants,
develop FBs that are not associated with MOs. spe-4 mutants
accumulate aberrant spermatocytes filled with distended MOs that are not
associated with FBs (Arduengo et al., 1998).

The spe-5
gene encodes a vacuolar ATPase B subunit (P. Hartley and S.W. L’Hernault,
unpublished) that localizes to the FB-MO (E. J. Gleason and S. W. L’Hernault,
unpublished). spe-5 mutants form FB-MOs that are greatly
distended and vacuolated. Usually,spe-5 null mutants
arrest as aberrant spermatocytes that contain four haploid nuclei (Figure 5B), but occasionally a few spe-5
self-progeny are observed so some functional spermatozoa must form (Machaca and L'Hernault, 1997). Because vacuolar ATPases acidify compartments
(Nishi and Forgac, 2002), the spe-5 defect
may reflect a lack of FB-MO acidification.

The spe-17 gene encodes a small soluble protein with no obvious conserved domains or
homologs outside nematodes (L'Hernault et al., 1993).
spe-17 mutants form FB-MOs with membrane-attached
ribosomes. The consequence is that spe-17 mutants retain
ribosomes in the spermatid. The FB can still disassemble and release MSP, but
many MOs do not fuse with the cell surface during spermiogenesis (Shakes and Ward, 1989). Motile spermatozoa can still form in
spe-17 mutants, and some are competent to engage in fertilization.

spe-10 mutants initiate FB-MO morphogenesis normally, but
the membrane surrounding the FB prematurely retracts so that FBs are left in the
residual body during spermatid budding. Budded spe-10
spermatids are deficient in MSP and resulting spe-10
spermatozoa are immotile and have vacuolated MOs that do not fuse with the
plasma membrane (Shakes and Ward, 1989). The
spe-10 gene encodes a four pass integral membrane protein
that contains a DHHC domain predicted to be a palmitoyl transferase that is localizes within FB-MOs (Gleason et al. 2006). The
presence of this motif implicates SPE-10 function in the posttranslational
palmitoylation of target proteins required for proper FB-MO function. In
addition to FB-MO defects, both spe-17 and spe-10 mutant males make spermatids that are only ~66% the wild-type size
and have nuclei that are eccentrically placed (Shakes and Ward, 1989).

fer-6 mutants makes spermatids in which the FBs detach from
the MO but fail to disassemble and release MSP. Many MOs fail to fuse with the
plasma membrane in fer-6 mutants, which form abnormal
spermatids (Ward et al., 1981).

fer-15 mutant spermatids fail to respond to in vitro activators and do not become spermatozoa (Figure 5; S. L’Hernault and S. Ward, unpublished observations).

fer-1 mutants have no apparent defects in the early stages
of FB-MO morphogenesis and have normal sized spermatids in which FBs disassemble
normally. However, the MOs fail to fuse with the plasma membrane, and
fer-1 mutants extend short pseudopods that do not allow
normal motility (Roberts and Ward, 1982; Ward et al., 1981). The fer-1 gene encodes a one pass transmembrane
protein in the dysferlin family with homology to at least seven human genes
(Achanzar and Ward, 1997), including one implicated in limb girdle
muscular dystrophy (Bashir et al., 1998). Human dysferlins are
probably involved in muscle membrane repair/remodeling (Bansal and Campbell, 2004), which is consistent with the fer-1
membrane fusion defect (Ward et al., 1981).

7. Cytoskeletal mutants

There are two spe genes that encode known cytoskeletal proteins.

The spe-26 gene encodes an actin binding protein, and mutants usually form aberrant
spermatocytes that do not become spermatids. Occasionally,
spe-26 mutants make spermatids that become spermatozoa, but
these spermatozoa do not participate in fertilization. A
spe-26 spermatocyte can complete meiosis and form four
haploid nuclei within a single, aberrant cell, but these cells sometimes contain
as many as 12 DAPI positive DNA regions (Varkey et al., 1995).

The spe-15
gene encodes a myosin VI that plays a sorting role as spermatids bud from the
residual body. spe-15 mutants partially fail in their
polarized delivery of mitochondria and FB-MOs to spermatids and actin filaments
and microtubules to the residual body. As a consequence, all of these cellular
components are distributed to both spermatids and residual bodies during
budding. When spe-15 spermatids are protease activated
in vitro, resulting spermatozoa usually lack normal
pseudopods (Kelleher et al., 2000).

spe-29 encodes a single pass transmembrane protein that is
only ~7.5 kD. This novel protein is predicted to be on the sperm surface (Nance et al., 2000).

Hermaphrodites homozygous for a mutation in any spe-8 pathway
gene accumulate spermatids that fail to activate into spermatozoa (Figure 5). Unlike most (> 40)
spermatogenesis genes, males homozygous for a mutation in any one of these four genes
produce spermatozoa that are competent for fertilization. The arrested spermatids
present in hermaphrodites that are mutant in the spe-8 pathway can
be activated to become spermatozoa if they are exposed to male-derived seminal fluid.
However, neither male nor hermaphrodite derived spermatids from either sex are entirely
normal; after protease treatment, mutant spermatids have an unusual spiky cytology,
whereas wild-type spermatids become cytologically normal spermatozoa (Minniti et al., 1996; Nance et al., 1999; Nance et al., 2000; Shakes and Ward, 1989).

Eighteen non-null spe-6 mutants allow partial bypass of any
spe-8 pathway mutant (spe-6 null mutants have
defects in FB-MO morphogenesis, see above). These non-null spe-6
mutations are scattered across the polypeptide sequence and some affect non-essential
residues in the kinase catalytic region. It is thought that SPE-6 negatively regulates
spermiogenesis until an activation signal is sent through the spe-8
pathway. Partial loss of SPE-6 function partly alleviates the need for
spe-8 pathway-mediated relief of SPE-6 negative regulation in
hermaphrodites (Muhlrad and Ward, 2002). These spe-6 suppressor mutants
have also been analyzed in a background that was not mutant for other genes in the
spe-8 pathway. In males, spe-6 suppressor
mutations show precocious activation of spermatids into spermatozoa. However, there is a
large reduction in spermatid numbers due to necrosis, and mutant males rarely sire
progeny after copulation. In contrast, hermaphrodite-derived spermatids show only
minimal signs of necrosis in spe-6 suppressor mutants (Muhlrad and Ward, 2002).

These data indicate that there are two spermiogenesis pathways, one active in males
and one active in hermaphrodites (Nance et al., 2000; Shakes, 1988). The pathway in hermaphrodites has two kinases (SPE-6 and SPE-8), one soluble
protein (SPE-27) and two transmembrane proteins (SPE-12 and SPE-29), suggesting it
functions in signal transduction. The physiological signal that initiates spermiogenesis
in hermaphrodites is not presently known. None of the genes that participate in the
spermiogenesis pathway for male-derived spermatids are presently known.

The spe-9 gene encodes a one-pass integral membrane protein with 10 EGF-like motifs, which
usually have extracellular functions such as adhesive and ligand-receptor
interactions (Singson et al., 1999). Transgenes bearing the
spe-9 coding sequence in which deletion or point mutations
were induced revealed that certain EGF repeats in the predicted SPE-9 protein
are more important than others for SPE-9 function during fertilization (Putiri et al., 2004).

The spe-41/trp-3 gene encodes a TRP calcium channel protein
that has a role in store-and, perhaps, receptor- operated calcium entry during
fertilization (Xu and Sternberg, 2003).

The spe-42 gene encodes a seven pass transmembrane protein, and two SPE-42 homologs are
evident in all multicellular animals with a complete genome sequence, including
C. elegans. In humans, one of these homologs has a
splice form expressed in testes, but the precise function of SPE-42 or any of
its homologs is not yet known (Kroft et al., 2005).

Spermatozoa derived from any of the seven mutants in this class have no detectable
motility defects, and they make contact with oocytes in the spermatheca. Male-derived
spermatozoa from five of these mutants (fer-14,
spe-9, spe-13,
spe-41/trp-3 and spe-42) render recipient,
formerly self-fertile, hermaphrodites sterile after mating; such hermaphrodites cease
laying embryos and begin to lay unfertilized oocytes (Singson et al., 1999; Xu and Sternberg, 2003). This sterility reflects the fact that
these mutant, male-derived spermatozoa can out-compete and functionally displace
endogenous, fertilization-competent hermaphrodite spermatozoa in the spermatheca (as in
Figure 5C). The consequence of this displacement is that oocytes
only contact fertilization defective spermatozoa during the narrow temporal window when
they are competent for fertilization.

SPE-41 (Xu and Sternberg, 2003), SPE-9 (Zannoni et al., 2003) and SPE-38 (Chatterjee et al., 2005) are all
plasma membrane proteins in spermatozoa. SPE-9 is also found in the plasma membrane of
spermatids, but SPE-41 and SPE-38 are found in the MOs and become plasma membrane
proteins when the MOs fuse during spermiogenesis. This observation places the SPE-41 TRP
channel function on the cell surface where it could participate in calcium fluxes
related to gamete fusion during fertilization. While SPE-41 is distributed on the plasma
membrane of both the cell body and the pseudopod, SPE-9 and SPE-38 localize to the
pseudopodial plasma membrane of spermatozoa (Figure 5C). These data
indicate that there are at least two ways for plasma membrane proteins required for
fertilization to reach the spermatozoan cell surface. Additional proteins important for
oocyte recognition, adhesion and/or fusion are likely to be encoded by
spe-13 and spe-36 and other sperm-expressed
genes that remain to be discovered.

10. Post-fertilization mutants

One mutant (spe-11) forms spermatozoa that are competent to
fertilize oocytes, but embryogenesis never begins properly and the defective embryo
always dies (Browning and Strome, 1996; Hill et al., 1989;
L'Hernault et al., 1988). After a wild type oocyte is fertilized by a
spe-11 spermatozoon, maternal meiosis is not usually completed
properly and a defective eggshell forms. While mitosis is observed in oocytes fertilized
by a spe-11 sperm, the spindle is not positioned correctly and
cytokinesis does not usually occur. The typical end result is that multiple nuclei share
a common cytoplasm in these defective embryos (Figure 5; Hill et al., 1989). Expression of the spe-11 gene normally occurs
only during spermatogenesis, and the SPE-11 protein has no clear homologs outside
nematodes (Browning and Strome, 1996). Ultrastructural examination of
spe-11 mutant spermatids reveals that they have defects in
perinuclear material surrounding the nucleus (Hill et al., 1989), which is
where the SPE-11 protein localizes in wild type spermatids (Browning and Strome, 1996). This sperm-derived material is apparently required for some as yet
unknown aspect of early embryogenesis. However, it does not have to be delivered via the
sperm because transgenic expression in the oocyte is sufficient to allow embryos that
have been fertilized with a spe-11 spermatozoon to survive
(Browning and Strome, 1996). Other work has shown that the C.
elegans sperm-derived nucleus (Sadler and Shakes, 2000) and
centriole (O'Connell, 2002; O'Connell et al., 2001) are
both required for embryogenesis. However, SPE-11 is the only known C.
elegans protein that is both required for embryogenesis and normally has a
sperm-specific expression pattern.

11. Future prospects

Progress in the study of C. elegans spermatogenesis has
accelerated in recent years as the molecular identities of many of the involved genes
have been discovered. The earliest stages of spermatogenesis employ genes
(cpb-1, ife-1, puf-8, spe-39 and
wee-1.3) that are not exclusively expressed during spermatogenesis.
If genes exist that regulate translational control or meiosis, but commence expression
after spermatogenesis has been initiated, they remain to be discovered. A partial
picture of the function of the FB-MO has emerged, but much work remains to be done. The
earliest stages of FB-MO morphogenesis are presumably controlled through the vesicular
trafficking pathway. These components are likely to be expressed outside the testes (as
is spe-39) and probably will require different types of mutant
screens and/or RNAi studies to be identified. Although the genetic control of
hermaphrodite spermiogenesis is becoming clearer, none of the genes that participate in
the predicted male spermiogenesis pathway have been identified. Mutants with specific
defects in male spermiogenesis are likely obtainable, but existing screening methods
that rely on recovering self-sterile hermaphrodites will not allow identification of
such mutants; new screens will need to be designed for this purpose. There has been much
recent progress in identifying and analyzing the sperm-expressed genes that participate
in fertilization. All five of the cloned genes that are important for fertilization
encode transmembrane proteins. How these transmembrane proteins organize to interact
with the egg during fertilization remains to be determined. Thespe-11 mutant reveals that sperm play a crucial role in the delivery of
components to the oocyte that allow it to progress through embryogenesis normally. It
will be interesting to learn if there are other proteins required for embryogenesis that
must be delivered by the sperm during fertilization.

12. Acknowledgements

I thank Sam Ward, Andrew Singson and members of the L’Herrnault lab for valuable
discussion. Work in my laboratory has been supported by grants from the NIH (GM040697) and NSF (IBN-0131532).

Machaca, K., and L'Hernault, S.W. (1997). The Caenorhabditis elegansspe-5 gene is required for morphogenesis of a sperm-specific organelle and is associated with an inherent cold-sensitive phenotype.
Genetics 146, 567–581.Abstract

Ward, S. (1986). The asymmetric localization of gene products during the development of Caenorhabditis elegans spermatozoa. In Gametogenesis and the Early Embryo, J. Gall, ed. (New York: A.R. Liss), pp. 55–75.